The junctional complex between plasma membrane (PM) and endoplasmic/sarcoplasmic reticulum (ERJSR) provides a structural framework for interaction of cell-surface and intracellular ionic channels. In skeletal muscle, the transverse tubular invagination of PM touches the terminal cistemae of SR and forms a close triad junction, which enables direct coupling between dihydropyridine receptors DHPRs) and ryanodine receptors (RyRs)-two key molecules involved in membrane excitation mediated release of intracellular Ca and muscle contraction (E-C coupling). In addition to the forward coupling from surface membrane depolarization to intracellular Ca release, a backward transmission process termed capacitative Ca entry serves to recharge the intracellular Ca stores after depletion of Ca from ER/SR, and plays important roles in a variety of cellular processes ranging from gene regulation to cell proliferation an poptosis. Evidence indicates that physical docking of ER with PM and conformation coupling from RyR to transient receptor potential proteins (TRPs) are involved in activation of the store-operated Ca channel (SOC), but the molecular signal that trigger opening of SOC remains largely unknown. Moreover, the role of SOC in muscle function has just begun to be realized. Junctophilins (JP) are a group of novel membrane proteins with a carboxyl-terminal hydrophobic segment that spans the ER/SR membrane and a cytoplasmic region containing repeated motifs that interact specifically with the PM, and may serve to anchor cell surface to intracellular membranes. The long term goal of this project is to understand the cellular and molecular function of these novel JP genes in the Ca signaling of muscle cells, specifically to test the hypothesis that JP plays important roles in the ultrastructural arrangement of the junctional membrane complex, and therefore may mediate the signal transduction process from surface membrane depolarization to intracellular Ca release (DHPR-about RyR), and from intracellular Ca depletion to store operated capacitative Ca entry (RyR-TRP). Combining the techniques of genetic manipulation of animal models, structural determination of the triad and dyad junction, functional examination of the E-C coupling unit, and reconstruction studies in cultured cells, the present project aims to test the cellular and molecular function of JPs in DHPR-mediated activation of the RyR/Ca release pathway (Aim 1), and to study the physiological function of JPs in RyR -mediated activation of the SOC entry pathway (Aim 2). This project represents the collaborative efforts of three laboratories, each with their distinct and complementary expertise on molecular cloning and gene expression, transgenic animal model and muscle physiology, electrophysiology and ion channel regulation, confocal microscopy and cellular Ca imaging. The ultimate goal of this team effort is to arrive at a fundamental understanding of JP nediated Ca signaling in the heart and skeletal muscle, and with the expectation that the studies with the mutant mice will provide further insights into the cellular mechanism of muscle exercise physiology and cardiovascular diseases.